Showing papers in "Journal of Microscopy in 1998"

3d microscopy of transparent objects using third-harmonic generation

Abstract: It is demonstrated that third-harmonic generation (THG) near interfaces in the refractive index or the third-order nonlinear susceptibility (chi(3)) permits three-dimensional imaging of transparent objects. The nonlinear dependence of THG on the excitation power provides inherent optical sectioning. At the same time, the nonresonant nature of THG, in combination with the near-IR excitation wavelengths used (1-2 µm), render this technique potentially (biologically) nondamaging and nonbleaching. A specific property of THG imaging is its sensitivity to - and potential use for imaging of - the relative orientation of interfaces with respect to the axis of propagation of the excitation radiation.

Aberration correction for confocal imaging in refractive‐index‐mismatched media

Abstract: Summary A major limitation to the use of confocal microscopes to image thick biological tissue lies in the dramatic reduction in both signal level and resolution when focusing deep into a refractive-index-mismatched specimen. This limitation may be overcome by measuring the wavefront aberration and pre-shaping the input beam so as to cancel the effects of aberration. We consider the images of planar and point objects in brightfield, single-photon fluorescence and two-photon fluorescence imaging. In all cases, the specimens are imaged using an oil-immersion objective through various thicknesses of water. The question of finite-sized pinhole is addressed and it is found, in general, that it is sufficient to correct only the first two or three orders of spherical aberration to restore adequate image signal level and optical resolution, at imaging depths of up to 50-100 wavelengths.

Optimal and near‐optimal filters in high‐resolution electron microscopy

Abstract: Two filters for improving the visibility of crystalline material in the presence of amorphous surface contamination layers in high-resolution electron microscope images can be constructed automatically from the information present in the Fourier transform of the recorded image The recorded signal is considered in the first approximation to be the sum of two signals which are uncorrelated in the frequency domain By estimating the power spectrum of the signal from the amorphous layer, an optimized estimate for the desired signal is given by the Wiener filter A second filter which uses the estimated amplitude of the amorphous signal to subtract out a background can be shown to be related to the Wiener filter The two filters are applied to an experimental image of zeolite and the effects of the two filters are compared

The Basics of Crystallography and Diffraction

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Contrast, resolution, pixelation, dynamic range and signal‐to‐noise ratio: fundamental limits to resolution in fluorescence light microscopy

Abstract: In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide-field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide-field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.

Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives

Abstract: The excitation efficiency in two-photon absorption (TPA) microscopy depends strongly — owing to the square dependence of the TPA fluorescence on the excitation intensity — on the temporal width of the excitation pulse. Because of their inherently large frequency bandwidth, ultrashort optical pulses tend to broaden substantially because of dispersion from propagation through the dispersive elements in the microscope. In this paper, the dispersion characteristics of a wide range of microscope objectives are investigated. It is shown that the induced dispersion can be pre-compensated in all cases for pulses as short as 15 fs. Because of the excellent agreement between the results from theoretical modelling and the experimental data, predictions of the possibility of dispersion control for microscope objectives in general, as well as for even shorter pulses, can be inferred. Since for TPA imaging the background due to single photon absorption processes and scattering is independent of the pulse width, proper dispersion pre-compensation — which minimizes the pulse duration at the focal point and hence maximizes the excitation efficiency — provides optimal image contrast in TPA microscopy.

Extended confocal microscopy of myocardial laminae and collagen network

Abstract: Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusion-stained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 μm in a contiguous mosaic across the surface. Approximately 50 μm was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 × 800 × 800 μm3 spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart.

Real time two-photon absorption microscopy using multi point excitation

Abstract: In this communication we present the development of a real time two-photon absorption microscope, based on parallel excitation with many foci. This pattern of foci is created by a two-dimensional microlens array. The fluorescence is detected by direct, non descanned detection on a CCD camera. Due to the parallel nature of both excitation and detection it is possible to speed up image acquisition significantly. This makes the instrument especially suitable for studying living specimens and/or real time processes. The optical design of the instrument is discussed and an imaging example is given. We specifically address the relation between the axial sectioning capability and the distance between the illumination foci at the sample.

Desktop X‐ray microscopy and microtomography

Abstract: Recent developments in X-ray microtomography have made it possible to miniaturize a CT scanner into a versatile and cost-effective desktop system that fits into any laboratory environment. The possibilities of the technique are demonstrated for a range of applications. It is also shown how an existing scanning electron microscope with an X-ray detector can, with a specially developed attachment, be transformed into an X-ray microscope and microtomograph.

High-precision distance measurements and volume-conserving segmentation of objects near and below the resolution limit in three-dimensional confocal fluorescence microscopy

Abstract: This study presents a method for high-precision distance measurements and for the volume-conserving segmentation of fluorescent objects with a size of the order of the microscopic observation volume. The segmentation was performed via a model-based approach, using an algorithm that was calibrated by the microscopic point spread function. Its performance was evaluated for three different fluorochromes using model images and fluorescent microspheres as test targets. The fundamental limits which the microscopic imaging process imposes on the accuracy of volume and distance measurements were evaluated in detail. A method for the calibration of the axial stepwidth of a confocal microscope is presented. The results suggest that in biological applications, 3D distances and radii of objects in cell nuclei can be determined with an accuracy of ≤ 60 nm. Using objects of different spectral signature, 3D distance measurements substantially below the lateral half width of the confocal point spread function are feasible. This is shown both theoretically and experimentally.

Global spatial sampling with isotropic virtual planes: estimators of length density and total length in thick, arbitrarily orientated sections

Abstract: Existing design-based direct length estimators require random rotation around at least one axis of the tissue specimen prior to sectioning to ensure isotropy of test probes. In some tissue it is, however, difficult or even impossible to define the region of interest, unless the tissue is sectioned in a specific, nonrandom orientation. Spatial uniform sampling with isotropic virtual planes circumvents the use of physically isotropic or vertical sections. The structure that is contained in a thick physical section is investigated with software-randomized isotropic virtual planes in volume probes in systematically sampled microscope fields using computer-assisted stereological analysis. A fixed volume of 3D space in each uniformly sampled field is probed with systematic random, isotropic virtual planes by a line that moves across the computer screen showing live video images of the microscope field when the test volume is scanned with a focal plane. The intersections between the linear structure and the virtual probes are counted with columns of two dimensional disectors. Global spatial sampling with sets of isotropic uniform random virtual planes provides a basis for length density estimates from a set of parallel physical sections of any orientation preferred by the investigator, i.e. the simplest sampling scheme in stereology. Additional virtues include optimal conditions for reducing the estimator variance, the possibility to estimate total length directly using a fractionator design and the potential to estimate efficiently the distribution of directions from a set of parallel physical sections with arbitrary orientation. Other implementations of the basic idea, systematic uniform sampling using probes that have total 3D × 4π freedom inside the section, and therefore independent of the position and the orientation of the physical section, are briefly discussed.

Theory of confocal fluorescence imaging in the programmable array microscope (PAM)

Abstract: The programmable array microscope (PAM) uses a spatial light modulator (SLM) to generate an arbitrary pattern of conjugate illumination and detection elements. The SLM dissects the fluorescent light imaged by the objective into a focal conjugate image, Ic, formed by the ‘in-focus’ light, and a nonconjugate image, Inc, formed by the ‘out-of-focus’ light. We discuss two different schemes for confocal imaging using the PAM. In the first, a grid of points is shifted to scan the complete image. The second, faster approach, uses a short tiled pseudorandom sequence of two-dimensional patterns. In the first case, Ic is analogous to a confocal image and Inc to a conventional image minus Ic. In the second case Ic and Inc are the sum and the difference, respectively, of a conventional and a confocal image. The pseudorandom sequence approach requires post-processing to retrieve the confocal part, but generates significantly higher signal levels for an equivalent integration time.

Why don't high‐resolution simulations and images match?

Abstract: Computer simulations have been used for many years to understand experimental high-resolution electron microscope images in a qualitative fashion, but the trend nowadays has been to attempt more quantitative image matching. This has led to the discovery that the contrast in experimental images is much less than in simulated images, typically by a factor of about three. There are many possible causes for this discrepancy, ranging from the mechanisms of scattering of electrons by the specimen through the calculations of the diffracted beam intensities and their focusing by the objective lens to the point spread function of the recording device. No single cause can explain all of the experimental contrast loss, although a combination of many factors could.

Multivariate statistical methods for the analysis of microscope image series: applications in materials science

Abstract: Multivariate data sets are now produced in several types of microscopy. Multivariate statistical methods are necessary in order to extract the useful information contained in such (image or spectrum) series. In this review, linear and nonlinear multivariate methods are described and illustrated with examples related both to the segmentation of microanalytical maps and to the study of variability in the images of unit cells in high-resolution transmission electron microscopy. Concerning linear multivariate statistical analysis, emphasis is put on the need to go beyond the classical orthogonal decomposition already routinely performed through principal components analysis or correspondence analysis. It is shown that oblique analysis is often necessary when quantitative results are expected. Concerning nonlinear multivariate analysis, several methods are first described for performing the mapping of data from a high-dimensional space to a space of lower dimensionality. Then, automatic classification methods are described. These methods, which range from classical methods (hard and fuzzy C-means) to neural networks through clustering methods which do not make assumptions concerning the shape of classes, can be used for multivariate image segmentation and image classification and averaging.

The coefficient of error of optical fractionator population size estimates: a computer simulation comparing three estimators

Abstract: The optical fractionator is a design-based two-stage systematic sampling method that is used to estimate the number of cells in a specified region of an organ when the population is too large to count exhaustively. The fractionator counts the cells found in optical disectors that have been systematically sampled in serial sections. Heretofore, evaluations of optical fractionator performance have been made by performing tests on actual tissue sections, but it is difficult to evaluate the coefficient of error (CE), i.e. the precision of a population size estimate, by using biological tissue samples because they do not permit a comparison of an estimated CE with the true CE. However, computer simulation does permit making such comparisons while avoiding the observational biases inherent in working with biological tissue. This study is the first instance in which computer simulation has been applied to population size estimation by the optical fractionator. We used computer simulation to evaluate the performance of three CE estimators. The estimated CEs were evaluated in tests of three types of non-random cell population distribution and one random cell population distribution. The non-random population distributions varied by differences in 'intensity', i.e. the expected cell counts per disector, according to both section and disector location within the section. Two distributions were sinusoidal and one was linearly increasing; in all three there was a six-fold difference between the high and low intensities. The sinusoidal distributions produced either a peak or a depression of cell intensity at the centre of the simulated region. The linear cell intensity gradually increased from the beginning to the end of the region that contained the cells. The random population distribution had a constant intensity over the region. A 'test condition' was defined by its population distribution, the period between consecutive sampled sections and the spacing between consecutive sampled disectors. There were 1000 independently simulated cell populations for each test condition, and a 'trial' was conducted for each of these cell populations. In each trial we calculated the (unique) true CE of the population size estimate and the three CE estimates obtained by applying the Scheaffer-Mendenhall-Ott (SMO) and both Gundersen-Jensen (GJ) estimators. We compared the estimated CEs with the true CEs for each population distribution. We found that the CE estimates obtained by the SMO estimator were closer to the true CEs and had less scatter than those of the nugget-modified GJ estimator. Both had small positive bias. The CE estimates obtained by the unmodified GJ estimator exhibited widely varying bias and large scatter. In all the population distributions we tested, the average true CE was very nearly proportional to 1/square root of QT, where QT is the average number of cells counted in the two-stage systematic sample.

On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device.

Abstract: High-pressure freezing (HPF) is currently the only method which enables adequate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and preparation methods, the technique has not yet reached its full potential. While suspensions can be prepared easily for HPF, tissue preparation is restricted by the need to compromise between different requirements and difficulties. (i) In order to achieve optimal freezing quality, very thin samples are required. (ii) There is mechanical difficulty in cutting such thin samples without distorting the organization of the tissue. (iii) The cutting and the succeeding preparation steps of small samples require long handling times (minutes), which may result in physiological and hence structural alterations. Computerized heat transfer simulations are presented which confirm that the efficiency of heat extraction from cylindrical samples contained within thin-walled metal tubes is higher than from standard flat discoid samples sandwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the quick excision of such cylinders of soft tissues. The device utilizes sharp gold needles of an inner diameter of 200 microm and wall thickness of 50 microm. The frozen sample contained in the soft gold needle permits all the manipulations needed for conventional cryo-preparation techniques for electron microscopy (e.g. cryo-sectioning, freeze-fracturing, freeze-substitution).

Accurate structure determination from image reconstruction in ADF STEM

Abstract: Annular dark-field (ADF) imaging in a scanning transmission electron microscope results in direct structure images of the atomic configuration of the specimen. Since such images are almost perfectly incoherent they can be treated as a convolution between a point-spread function, which is simply the intensity of the illuminating electron probe, and a sharply peaked object function that represents the projected structure of the specimen. Knowledge of the object function for an image region of perfect crystal allows the point-spread function to be directly determined for that image. We examine how the object function for an image can then be reconstructed using a Wiener filter, the CLEAN algorithm and a maximum entropy reconstruction. Prior information is required to perform a reconstruction, and we discuss what nature of prior information is suitable for ADF imaging.

Elongate cavities and skin–core structure in Nephila spider silk observed by electron microscopy

Abstract: Major ampullate silk fibres from the orb-weaving spider Nephila madagascariensis were analysed by transmission electron microscopy. The fibres have a thin outer layer surrounding a column of apparently homogeneous material which contains elongate cavities orientated parallel to the silk fibre axis. The cavities appear similar to ‘elongate vacuolar droplets’ observed in the silk of Antheraea silkmoth larvae. The overall skin–core structure is probably the result of a rheological pattern originating in the two secreting regions recognized in Nephila silk glands; the cavities indicate material inhomogeneities.

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages.

Abstract: Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented. AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.

High‐pressure freezing for immunocytochemistry

Abstract: Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross-linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample-stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well-frozen material obtainable to approximately 15 microm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystal-artefact-free freezing of samples up to 200 microm in thickness. There have been few reports of high-pressure freezing for immunocytochemical studies and there is no consensus on the choice of post-freezing sample preparation. A range of freeze-substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin-embedding, polymerization and immunocytochemical labelling. Freeze-substitution in acetone containing 2% osmium tetroxide followed by epoxy-resin embedding at room temperature gave optimum morphology. Freeze-substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl-embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze-substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice-crystal damage in sections of embedded material. Thus a slow warm-up to freeze-substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze-substitution media from immunocytochemical labelling experiments.

Improved staining of F-actin and co-localization of mitochondria in plant cells

Abstract: Permeating the cell membrane with glycerol instead of detergents (e.g. NP-40) and/or organic solvents (e.g. DMSO) significantly improves the visualization of F-actin with rhodamine phalloidin. In particular, the thinnest bundles are more intensely stained, better delineated and observation time is prolonged dramatically. Furthermore, the method is gentle to membranous organelles, which makes, for example, co-localization of mitochondria and actin possible.

Star length distribution: a volume-based concept for the characterization of structural anisotropy.

Abstract: Determination and quantification of anisotropy is of great interest in research fields dealing with physical structures or surface textures. In this paper, a volume-based method is presented, which essentially determines the mean object length in a certain direction for a typical point within a structure or texture. The mean object lengths for all orientations together form the so-called star length distribution (SLD). The validity and the accuracy of the SLD method are investigated, and illustrated by applying it to trabecular bone. By using a line sampling algorithm, the relation with other anisotropy measures could be studied analytically. Preliminary tests suggest that with SLD a more exact description of the mechanical properties of porous structures may be obtained than with other anisotropy measures. However, due to possible secondary orientations that become apparent with SLD, a fabric tensor must be of rank higher than two in order to properly describe an orthogonal structure mathematically.

HREM and STEM of intergranular films at zinc oxide varistor grain boundaries

Abstract: Grain boundaries in model ZnO–Bi2O3 and ZnO–Bi2O3–CoO varistors and a commercial multicomponent varistor have been characterized by high-resolution electron microscopy (HREM) and scanning transmission electron microscopy (STEM), in order to determine the relationship between Bi grain boundary segregation and formation of thin intergranular films. By controlling Bi2O3 content, applied pressure and temperature, the grain boundary Bi excess has been systematically varied from nearly zero to ΓBi = 1 × 1015 cm−2 (≈ 1 monolayer), as measured by HB 603 STEM using an area-scan method. HREM shows that intergranular amorphous films are clearly distinguishable in samples with ΓBi > 8 × 1014 cm−2. These films range in thickness, depending on the Bi excess, from 0.6 to 1.5 nm. Similar films of ≈ 1 nm thickness are widely observed in the commercial varistor. The composition of the films is a ZnO–Bi2O3 solid solution, which is in all cases more enriched in ZnO than the bulk eutectic liquid. The Bi-doped grain boundaries in ZnO varistors therefore contain an intergranular amorphous film which has not only an equilibrium thickness, but also a distinct equilibrium composition.

Estimating surface area by the isotropic fakir method from thick slices cut in an arbitrary direction

Abstract: The proposed fakir method for estimating surface area is based on counting the intersections between the surface lying within a thick slice, and an isotropic spatial grid consisting of a combination of linear probes called fakir probes. An unbiased procedure using a directly randomized spatial grid rather than sections with randomized directions is presented. The method is applicable if perfectly registered serial sections of the surface are available in a thick slice while the direction of the slice can be arbitrary. The efficiency of the fakir method using different arrangements of orthogonal triplets of fakir probes is evaluated and it is shown that mutually shifted probes are superior to non-shifted ones. The application software for interactive counting of intersections between computer-generated fakir probes and the surface within the stack of digitized images is described and demonstrated by two examples: estimation of the surface area of individual tobacco cell chains using a confocal microscope, and estimation of the total area of exposed surface of mesophyll cells in a barley leaf using a wide-field transmission microscope.

Quantitative analysis of valence electron energy‐loss spectra of aluminium nitride

Abstract: The optical properties and electronic structure of aluminium nitride are determined using valence electron energy-loss spectroscopy in a dedicated scanning transmission electron microscope. Quantitative analysis of the experimental valence electron energy-loss spectra to determine the electronic structure encompasses single scattering deconvolution of the valence electron energy-loss spectra to calculate the energy-loss function, Kramers-Kronig analysis of the energy-loss function to reveal the complex dielectric function, transformation of the dielectric function into the optical interband transition strength via optical property relations and finally critical-point analysis of the interband transition strength. The influence of both experimental and analytical parameters on the final result was studied systematically to define and improve the understanding of the methods. To check the reliability of this technique the interband transition strength determined was compared with results of vacuum ultraviolet spectroscopy. Good agreement was found if sample preparation was taken into account. The preparation of the specimen for the transmission electron microscopy has an effect on the electronic structure. Quantitative analysis of valence electron energy-loss spectroscopy, using the methods presented, is an important and capable method to determine the electronic structure of materials and it has the benefit of high spatial resolution.

Iterative structure retrieval techniques in HREM: a comparative study and a modular program package

Abstract: A retrieval technique for crystal structures using high-resolution electron microscopy is presented. The inversion of the complex structure-to-image relation is performed by numerical optimization of the configuration space of object models. Unlike structure refinement in X-ray crystallography, the method operates on unknown defect structures on a nanometre scale. The diversity of crystal defects examined and the differences in microscope types and alignment conditions makes it necessary to adapt the basic algorithm to a broad variety of needs resulting in a modular program package for general use. New developments, such as a real space slice function calculation, problem adapted optimization strategies, and finally an examination of iterative matching of image series and exit wavefunctions are presented.

Beam damage of polypropylene in the environmental scanning electron microscope: an FTIR study

Abstract: The environmental scanning electron microscope allows the examination of virtually any specimen in a gaseous environment without the need for coating or drying. Experimental evidence, however, suggests that significant electron beam damage occurs in hydrated specimens. It is thought that water molecules, ionized by the electron beam, produce hydrogen and hydroxyl free radicals which attack the organic material of the sample. In order to elucidate the beam damage mechanisms, areas of polypropylene films were exposed to the electron beam at varying doses and exposure times under both hydrating and dehydrating conditions. The chemical changes occurring as a result of electron-beam irradiation were determined using Fourier transform infra-red microscopy. Direct interaction of the electron beam with the polymer results in extensive cross-linking. In the presence of water, free-radical-initiated reactions lead to hydrolysis and oxidation of the polymer.

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity‐modulated multiple‐wavelength scanning (IMS) technique

Abstract: We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.

Electron beam‐induced changes in vitreous sections of biological samples

Abstract: Nonpretreated high pressure frozen samples of Zea mays, cartilage and human erythrocytes were cryosectioned and observed at 110 K in a cryoelectron microscope. Changes induced by medium doses of electron irradiation (< 10 ke nm−2) are described. After some ke nm−2, the most conspicuous cutting artefacts are erased to a large extent and the visibility of the cell organelles is improved. The sections, compressed in the cutting direction by the sectioning process, shrink once more, in the same direction, when irradiated. This shrinkage depends on the section support and on how the section is adsorbed to it. Shrinkage is not uniform; it is most pronounced in mitochondria, condensed chromatin and nucleolus. This differential shrinkage improves the visibility of major structures on the section and, as a result, ‘nicer’ images are recorded. However, this apparent improvement is a beam-induced artefact that must be paired with a loss of high resolution information.